Light and shade in the photocontrol of Arabidopsis growth

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Light and shade in the photocontrol of Arabidopsis growth Giorgio Morelli and Ida Ruberti Plants have evolved sophisticated sensing mechanisms that operate through phytochromes, perceiving changes in the red:far-red ratio, which trigger morphological changes to avoid shade. The shade-avoidance response essentially redirects resources and growth potential from the leaf and storage organs into increased extension growth to optimize light capture by plants. Recent studies implicate ATHB-2, a homeodomain-leucine zipper transcription factor, as a regulator of shade-avoidance responses and establish a strong link between this factor and auxin signaling. The action of ATHB-2 is likely to generate changes in auxin distribution that produce distinct but coordinated effects on different cell types across the plant. Future studies should highlight how polarity of auxin transport is altered in response to light-quality changes. Published online: 12 August 2002

Giorgio Morelli Unità di Nutrizione Sperimentale, Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione, Via Ardeatina 546, 00178 Rome, Italy. Ida Ruberti Centro di studio per gli Acidi Nucleici, c/o Dip. di Genetica e Biologia Molecolare, Università di Roma La Sapienza, P.le Aldo Moro 5, 00185 Rome, Italy. e-mail: [email protected]

As sessile organisms, plants have evolved sophisticated mechanisms to perceive environmental cues and adapt their developmental pattern to changes in the natural environment, thereby ensuring survival and reproduction. Being photosynthetic, plants are especially sensitive to their light environment. Using multiple photoreceptors absorbing in the ultraviolet, blue and red–far-red (R–FR) spectral ranges, plants constantly monitor light intensity, quality and duration to control diverse developmental processes. Whenever plants grow in close proximity, there is competition for light. As a plant canopy grows and fills up space, a reduction in the ratio of R:FR light occurs because FR light is filtered through or reflected by neighbouring vegetation. Plants have evolved two opposing strategies in response to competition for light: shade tolerance and shade avoidance. Angiosperms have an impressive capacity to avoid shade. They perceive the R:FR ratio as an accurate indicator of neighbour proximity through the phytochrome photoreception system, which triggers morphological changes even before plants are directly shaded. Responses to shade are many and varied. The most dramatic shade avoidance response is the stimulation of elongation growth. This response is remarkably rapid and is reversible. In dicot plants, elongation growth induced by low R:FR ratios is often associated with a reduction of leaf development, a marked strengthening of apical dominance, and reduction in branching. Moreover, important responses to canopy shade are acceleration of flowering and reduced resources for storage and reproduction. This is associated with reduced seed set, truncated fruit development, and often a reduction in the germination ability of the seeds produced [1–4] (Fig. 1). http://plants.trends.com

Multiple phytochromes contribute to Arabidopsis shade avoidance responses

In Arabidopsis there are five phytochromes named PHYA–PHYE: they absorb mainly R–FR light. PHYA also responds to broad-spectrum light (UV-A to FR) of very low intensity [5,6]. PHYA is light-labile and its synthesis is down-regulated in the light. Consequently, phytochrome A is present at high concentrations in etiolated seedlings but at much lower levels in green plants. PHYB– E are light stable and present throughout the plant life cycle [7]. phyA mutants are essentially blind to FR light, suggesting a role for PHYA in seedling development under dense canopies. Consistently, it has been observed that de-etiolation of phyA mutants is severely impaired compared with wild-type plants if grown in deep-canopy shade, which leads to premature death [8]. By contrast, PHYB mediates de-etiolation in response to R light, complementing the FR response of PHYA to promote de-etiolation of emerging seedlings over a wide range of R:FR environments, from canopy shade to direct sunlight. As phytochrome A declines in photoautotrophic seedlings, the role of light-stable phytochromes becomes predominant in perceiving the R:FR light ratio. In spite of initial suggestions that shade avoidance responses are triggered by a single phytochrome, the study of Arabidopsis mutants has revealed that multiple phytochromes are involved. The phyB mutant constitutively displays shade avoidance traits such as elongated hypocotyl, stem, petioles and leaves, acceleration of flowering and higher apical dominance under high R:FR conditions. However, phyB null mutants also show typical shade avoidance responses under low R:FR conditions, indicating that other phytochromes also play a role in mediating these responses [2]. phyD and phyE single mutants are essentially indistinguishable from wild-type plants. However, phyB phyE, and, to a lesser extent, phyB phyD double mutants had longer petioles and flower earlier than phyB mutants. This led to the proposal that in conjunction with PHYB, PHYD and PHYE function in the regulation of shade avoidance responses [9–11]. No mutations in PHYC have been identified yet, but overexpression studies suggest a role in primary leaf expansion [7]. Genetic and molecular approaches have identified two classes of signaling components, those

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Cross-talk between light and hormone signaling

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Fig. 1. Effect of shade light on plant development. Plants grown under high (a) or low (b) red:far-red (R:FR) ratios. The two forms of phytochrome are depicted in the middle. PR, the R-light-absorbing form is converted by R light to the FR-light-absorbing form called PFR. The PFR form, in turn, can be converted back to PR by FR light.

acting downstream of a single photoreceptor and those acting downstream of multiple photoreceptors. The class that acts downstream of multiple photoreceptors includes both positive (i.e. HY5) and negative (DET/COP/FUS) regulators of photomorphogenesis. The study of mutants with phenotypes under specific light conditions and of PHY-interacting proteins revealed a complex signaling web with both nuclear and cytoplasmic factors. The biochemical function of most of these factors, as well as their position in the PHY signaling pathways, are largely unknown, and, thus, a clear picture of phytochrome signal transduction has not yet emerged [7,12]. However, a recent key discovery suggests that an important form of phytochrome regulation of gene expression is the direct interaction of activated phytochrome with sequence-specific DNA-binding proteins in the nucleus [13,14]. http://plants.trends.com

Several studies have suggested that auxin, brassinosteroids (BRs) and gibberellin (GAs) are all involved in the regulation of photomorphogenic processes. These hormones also influence plant stature and organ size, regulating cell division and/or cell expansion [7]. Auxin is involved in diverse developmental processes including cell enlargement, vascular tissue differentiation, root initiation, gravitropic and phototropic responses, and apical dominance. Indole-3-acetic acid (IAA) is synthesized in young leaves of the shoot and transported downward to the root tip through the vasculature. IAA is also synthesized in other plant organs such as cotyledons, expanding leaves and roots [15]. It has been suggested that auxin might act as a coordinator of growth because it is the only hormone transported polarly along the plant [16,17]. Recently, it has been shown that polar auxin transport (PAT) is affected in a light-dependent manner [18], and an increasing body of evidence suggests intimate interactions between phytochrome and auxin signaling. Several links between light and auxin have been defined using primary auxin-response genes as genetic and molecular tools. These genes comprise three major classes known as the Aux/IAA, GH3-like and SAUR gene families [19]. Aux/IAA proteins appear to be central to the auxin response. They mediate downstream responses by regulating gene activity through specific interactions with members of the auxin response factor (ARF) family [20]. ARF proteins contain a highly conserved DNA-binding domain that binds to specific DNA sequence motifs present in the promoters of many auxin-regulated genes [21,22]. The study of several aux/iaa mutants has established a strong link between auxin signaling and photomorphogenesis. A dominant mutation in SHY2/IAA3 acts as a suppressor of the long-hypocotyl phenotype of mutants with reduced levels of all phytochromes [23], or a null allele of PHYB [24]. Mutations in two other AUX/IAA genes, AXR2/IAA7 and AXR3/IAA17, lead to alteration in subsets of phytochrome responses [25]. Moreover, the auxin-insensitive mutants msg1/nph4/arf7 and msg2/iaa19 exhibit decreased phototropism as well as gravitropism in hypocotyls. msg1/nph4/arf also exhibits a reduced auxin induction of auxinregulated genes such as GH3, SAUR and several Aux/IAA genes [26,27]. Interestingly, a recombinant oat PHYA can phosphorylate SHY2/IAA3, AXR3/IAA17, IAA1, IAA9 and Ps-IAA4 in vitro [28]. Thus, one possible mechanism by which light could affect the auxin-response pathway(s) is through the direct regulation of Aux/IAA protein activity.

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Fig. 2. Dendrogram showing the order of the pairwise alignment of the Arabidopsis HD-ZIP II proteins. The distance along the horizontal axis is proportional to the differences between sequences; the distance along the vertical axis has no significance. The alignment was made with the entire coding sequences. The genes affected and unaffected by lightquality changes are indicated in red and blue, respectively. The response of the other HD-ZIP II genes (indicated in black) to changes in the R:FR ratio has not been determined, yet. ATHB-2 (EMBL Data Library Accession no. X68145), ATHB-4 (Y09582), ATHB-17 (AJ431181), HAT1 (U09333), HAT2 (AJ431183), HAT3 (U09338), HAT9 (U09341), HAT14 (AJ431182), HAT22 (U09336).

Analysis of members of the GH3-like gene family has also suggested a cross-talk between auxin response and light regulation. For instance, loss-of-function mutations in the auxin-inducible FIN219 gene affect the FR-mediated inhibition of hypocotyl elongation regulated by PHYA [29]. Furthermore, a dominant mutation in another GH3-like gene results in a dwarf phenotype in light, but not in darkness [30]. Conversely, the yucca mutant, containing elevated levels of auxin, is taller than the wild type under white light but shorter in darkness [31]. Altogether, these data point to a role for Aux/IAA and GH3-like gene products in the regulation of light-dependent symmetrical and/or differential cell elongation, at least in the hypocotyl. BRs are required for skotomorphogenic development – the elongation of hypocotyls and epicotyls in darkness [32,33]. BRs might also be required for polar elongation of leaf cells, as suggested by the finding that the Arabidopsis ROTUNDIFOLIA3 gene encodes a cytochrome (CY) P-450 protein homologous to C-22 and C-23 steroid hydroxylases involved in brassinolide biosynthesis [34]. http://plants.trends.com

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Recent reports have suggested a strong link between BR metabolism and phytochrome signaling. In pea, a small G protein belonging to the Ras superfamily named PRA2 and DDWF1, a CYP450 steroid C-2 hydrolase that catalyses a step of brassinosteroid synthesis, were found to interact in a two-hybrid assay and co-localize in the endoplasmic reticulum, strongly suggesting that these proteins act together [35]. Biochemical data indicated that PRA2 specifically regulates the activity of DDFW1. Moreover, both genes were shown to be repressed by light and predominantly expressed in the rapidly elongating zone of etiolated pea epicotyls [35,36]. Altogether, the data suggested that PRA2, in combination with DDWF1, might work as a light switch to decrease the rate of cell elongation during the de-etiolation process. There is evidence to suggest that BRs also have a role in the regulation of hypocotyl elongation of plants growing in a natural day–night cycle. The enhanced expression of CYP450 in the Arabidopsis bas1-D mutant suppresses the long-hypocotyl phenotype of the phyB-4 mutant [37]. Accordingly, transgenic Arabidopsis plants overexpressing DDWF1 exhibit enhanced hypocotyl growth in the light [35]. Finally, the Arabidopsis dwf1-10 mutant, an allele of the DIMINUTO gene encoding a FAD-dependent oxidase involved in BR biosynthesis, does not elongate the hypocotyls in canopy shade light [38]. Interestingly, BRs have also been shown to act synergistically in several responses controlled by auxin [39], and to induce the auxin-regulated gene SAUR-AC1 [40]. Like auxin, gibberellins are involved in diverse developmental processes. In Arabidopsis and rice, bioactive GA appears to control the amount of growth by repressing the action of negative regulatory proteins in a dose-dependent manner [41]. Therefore, a fine-tuned regulation of GA biosynthesis and metabolism is likely to play a crucial role. Several pieces of evidence have suggested that GA biosynthesis is regulated by the phytochrome system during seed germination, seedling growth and photoperiodic induction of flowering [42]. Of particular interest is the finding that the pea LE gene, encoding the enzyme catalysing the step GA20 to GA1, is regulated by auxin [43]. Because application of PAT inhibitors just below the apical bud of intact plants or decapitation markedly reduces the amount of GA1, it is suggested that, at least in pea, IAA can promote elongation growth partly through the regulation of GA1 biosynthesis [44]. In conclusion, research on auxin, BR and GA has uncovered fascinating interactions between these crucial regulatory molecules, and cross-talk between hormone and light signaling pathways. However, the integration of these distinct signals in the regulation of shade-induced elongation is largely unknown.

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Fig. 3. Working model depicting basipetal auxin transport under daylight and shade. In daylight (a), auxin synthesized in the shoot is transported to the root mainly through the central cylinder (indicated as central route). By contrast, in the shade (b), the outer cell layers (indicated as lateral route) contribute most significantly to the indole-3-acetic acid (IAA) movement towards the root. However, the lateral route is less effective in drawing out auxin from the auxin source. Thus, auxin levels increase in cell layers external to the central cylinder in leaves and stem. This redistribution of auxin in the aerial part of the plant reduces cell expansion in leaves and enhances cell elongation in the stem. Furthermore, the higher lateral transport of auxin results in a net reduction of auxin transported through the developing vascular system, which produces a let up in vascular differentiation and a decrease in the auxin concentration reaching the root. The inset represents a magnification of the region connecting the stem and leaves. There, the phytochrome system could be acting as a control gate directing auxin to the central route in the daylight (a), and to the lateral route in the shade (b). Elongation responses elicited by plants in open canopies can be reduced by blinding the stems to reflected far-red (FR) light using appropriate filters [3]. Peanut shapes in the lateral and central route specify auxin; PAT-taxicabs indicate the polar auxin transport system.

Mechanisms that might control shade avoidance responses Early intermediates in shade signaling

To date, only two genes [ATHB-2 (also known as HAT4) and ATHB-4] that are specifically regulated by light-quality changes have been identified [45–47]. ATHB-2 and -4 are members of a large class of Arabidopsis proteins characterized by the presence of a homeodomain-leucine zipper motif (HD-Zip) unique http://plants.trends.com

to higher plants [45]. HD-Zip proteins interact with DNA and act as positive (i.e. ATHB-1) and negative (i.e. ATHB-2) regulators of gene expression [48–50]. The HD-Zip transcription factors have been grouped into four different families, namely HD-ZIP I, II, III and IV. Interestingly, ATHB-2 and -4 encode proteins of the same family (HD-ZIP II, Fig. 2). However, at least one member of the HD-ZIP II family, HAT22, is not regulated by light-quality changes [51]. The light regulation of ATHB-2 is complex, involving at least three distinct phytochromes. In etiolated seedlings, the gene is expressed at relatively high levels and is rapidly down-regulated by FR or R light through the action of PHYA and a phytochrome other than B, respectively. In young seedlings and mature plants, ATHB-2 is rapidly induced by low R:FR ratios, largely through the action of a phytochrome other than A and B and secondarily by phytochrome B [52]. Recently, the finding that ATHB-2 has a negative auto-regulatory loop suggested an effective mechanism for maintaining a tight control of the ATHB-2 concentration in the nuclei, both under high and low R:FR ratios. It has also been suggested that ATHB-2 might be involved in a complicated regulatory network involving HD-ZIP II genes, similar to the networks found in animal homeobox genes. HD-ZIP II proteins might recognize similar target sequences because amino acid sequences are highly conserved in their HD-Zip domains. If this is the case, both autonomous and mutual regulation are to be expected among the HD-ZIP II genes. In shade light, it might be important that ATHB-2 downregulates other HD-ZIP II genes to exclusively regulate common target genes [53]. The involvement of ATHB-2 in shade avoidance responses has been corroborated by alterations in development observed in seedlings with increased or reduced ATHB-2 expression. Seedlings overproducing ATHB-2 had phenotypes reminiscent of those displayed by wild-type plants grown in FR-rich light. Conversely, seedlings with reduced levels of ATHB-2 had reciprocal phenotypes [50]. Studies in the hypocotyl of these transgenic plants have indicated that the alteration of elongation growth was the result of changes in both cell expansion and cell proliferation. Plants with reduced levels of ATHB-2 showed shorter epidermal and cortical cells and increased proliferation of secondary vascular tissue. By contrast, the elongated phenotype in the ATHB-2 overexpressing plants was shown to be the consequence of the same two events but in the opposite direction. Similar changes have been observed in wildtype seedlings grown in environmental conditions simulating canopy shade, indicating that low R:FR ratios produce distinct but coordinated effects on different cell types through the action of ATHB-2 [50]. A working model for shade-induced responses

Consistent with the role of auxin transport direction changes in elongation growth responses to directional light sources and alterations in the perceived gravity

Review

Acknowledgements We are grateful to Franco Marchiolli for the preparation of the figures. Our apologies to the many researchers whose work or original publications could not be cited here because of space limitations. This work was supported in part by the European Union Life Science Programme (contract no. QLG2-CT-1999–00876 to I.R.), by the Consiglio Nazionale delle Ricerche Target Project on Biotechnology (to G.M. and I.R.) and by the Ministero Istruzione, Università e RicercaConsiglio Nazionale delle Ricerche Strategic Project on Biotechnology (to G.M. and I.R.).

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vector [54], it has been found that auxin and the PAT system are important components of the elongation process induced by shade [4,50]. In the case of tropic responses, lateral transport of auxin across gravity or light-stimulated plant tissues drives differential growth [54]. By analogy, it was proposed that extension growth phenomena induced by neighbour detection and shade are the result of a laterally symmetric redistribution of auxin regulated by ATHB-2 [4,50]. That is, low R:FR ratios might promote elongation in the hypocotyl and petioles and limit cell expansion in cotyledons and leaves by altering auxin transport in the seedling (Fig. 3). Recent studies have found a correlation between endogenous auxin levels and hypocotyl length [31,55,56]. Moreover, several data support the hypothesis that an optimal IAA concentration is required for cotyledons and leaves to expand correctly [15]. For example, mutants with increased IAA levels, such as superroot1 and 2, show a reduced leaf expansion rate [15]. By contrast, gain-of-function mutations in the AUX/IAA genes SHY2/IAA3, AXR2/IAA7 and AXR3/IAA17 promote cotyledon expansion and development of leaves in etiolated seedlings [20]. The dark-to-light transition of the seedlings results in the activation of PAT systems in different cell types. This activation is likely to produce a transient reduction of the auxin pool which, in turn, triggers the initial expansion of cotyledons and inhibits hypocotyl elongation. It has been proposed that exposure of photoautotrophic seedlings to shade light produces a reduction of PAT through the central cylinder [50]. This, in turn, is likely to result in a transient increase of the auxin pool because the outer cell layers are significantly less efficient in draining out auxin from the auxin source (Fig. 3). A reduction of PAT through the central cylinder is also consistent with the inhibition of secondary vascular growth observed in the hypocotyls of seedlings expressing high levels of ATHB-2 as well as in wild-type seedlings grown in FR-rich light [4,50]. Auxin efflux and influx regulators are likely to play a major role in the laterally symmetric redistribution of auxin, and a recent finding strongly supports this view. Klaus Palme and co-workers have identified PIN3, a regulator of auxin efflux, as a component of the lateral auxin transport system regulating tropic growth. In addition, the finding that PIN3 relocalizes in response to gravity has provided a mechanism for redirecting auxin flux to trigger asymmetric growth [57]. The decreased PAT in the central cylinder should also produce a decrease in the auxin concentration reaching the root, resulting in a reduction of lateral root formation and slower growth of the main root. The root phenotype of ATHB-2 overexpressing seedlings supports this hypothesis. Primary root growth, lateral root formation and secondary vascular growth are all inhibited by elevated levels of ATHB-2, and at least the lateral root phenotype is rescued by exogenous IAA [50]. Moreover, recessive mutations in TIR3, a gene required http://plants.trends.com

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for normal PAT from the shoot into the root, result in the near absence of lateral roots, which is rescued by exogenous IAA [58]. Recently, the cloning of TIR3, renamed BIG, has provided further evidence that this gene has a role in PAT. BIG encodes an enormous protein with significant identity to the Drosophila protein Calossin, which is involved in the control of synaptic transmission at neuromuscular junctions [40]. Like synaptic transmission, the asymmetric distribution of auxin efflux carriers at the plasma membrane depends on targeted vesicle transport [59]. Thus, it has been hypothesized that BIG is involved in proper positioning of auxin efflux carriers at the plasma membrane. Consistently, treating tir3 seedlings with inhibitors of PAT changed the intracellular localization of the auxin efflux carrier PIN1 [40]. Finally, recent molecular data further support the model. Microarray experiments showed a significant increase in the level of several IAA mRNAs (IAA2, IAA7, IAA19) in wild-type plants after exposure to FR-rich-light illumination for 4 h (Arabidopsis Functional Genomic Consortium, Experiments 8130, 8266, 20902, http://genome-www5.stanford.edu/ MicroArray/SMD/). In addition, auxin levels were indirectly visualized in seedlings exposed to shade light using the synthetic DR5::GUS auxin reporter [60], whose activity correlates with direct auxin measurements [61]. Consistent with the model, GUS expression was significantly increased in cotyledons and in the hypocotyl of DR5::GUS seedlings exposed to 4-h-FR-rich-light illumination. Remarkably, crosssections of hypocotyls revealed the GUS staining in the outer cell layers (M. Carabelli et al., unpublished). Perspectives

In the past few years, there have been important advances in our understanding of shade avoidance responses in plants. The involvement of multiple phytochromes in these responses has been established, specific HD-Zip transcription factors have been identified as downstream components of the phytochrome signaling pathways, and auxin has been implicated as a coordinating signal in the overall plant response to shade light. We have summarized the first experimental evidence to provide insights into the regulation of shade avoidance responses by changes in auxin distribution. Future studies should examine how polarity of auxin transport is controlled and altered in response to shade light, and investigate the regulatory role of ATHB-2 and related proteins in this process. In addition, experiments should be designed to find out how distinct hormone signaling pathways are integrated in the control of elongation growth. Finally, shade-induced responses, providing strong and easily scorable phenotypes, might serve as useful assays for future physiological and genetic investigations. These assays should greatly assist dissection of the complex mechanism that continuously monitors the environment and coordinates morphogenesis throughout the plant.

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